Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection

Ecdysone triggered PGRP‐LC expression controls Drosophila innate immunity

Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP‐LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid‐regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP‐LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals

Drosophila Model of Leishmania amazonensis Infection

This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades

Plasmatocytes from adult flies phagocytose and control <i>Leishmania</i> parasites.

<p>DsRed-expressing amastigotes were injected alone or with polystyrene beads in <i>Hml(Δ)</i>Gal4-eGFP flies. At indicated times, hemocytes were harvested from the hemolymph and analyzed by confocal microscopy. (A) In eGFP-expressing hemocytes from flies injected with polystyrene beads and amastigotes, the cells contained large amounts of intracellular beads (blue) and just one intracellular parasite (red) confirming the inhibitory effect of beads in phagocytosis. (B) 24 h post-injection, plasmatocytes from flies injected only with parasites contained several amastigote forms, while at 72 h after infection some amastigotes differentiated to the long and flagellated promastigote form and were observed inside plasmatocytes (arrow in (C)). (D) Phagocytosis blockage by injection of polystyrene beads increased the parasite burden of flies over time. The control group, flies not injected with beads, prevented parasite growth without completely clearing infection. Parasite burdens were assayed by limiting dilution of single flies, with a minimum of 12 animals per time point. Whiskers represent 10%-90% limits of the sample. Statistical significance was determined by two-way ANOVA.</p

CD36 concentrates at the PV membrane juxtaposed to parasites.

<p>293T cells coexpressing mCherry-Rab7a and mCerulean3-CD36 were infected with GFP-expressing parasites and analyzed by confocal microscopy. (A) In the first hour of infection, CD36 was mainly co-localized with Rab7a in positive cytoplasmic vesicles, many of which were adjacent to the PV, and at cell membranes (arrowheads) and the entire PV. (B) By 6 h post-infection, CD36 was highly concentrated at the PV membrane juxtaposed to the amastigotes (arrows) while Rab7a was evenly distributed. (C) Amastigotes harvested from mCherry-CD36 expressing 293T cells by mechanical disruption retained CD36 (red) at the posterior pole indicating a stable interaction 24 h after infection.</p

In macrophages, CD36 recruitment is delayed in PVs containing <i>L</i>. <i>amazonensis</i> promastigotes.

<p>(A) PV-containing promastigotes did not exhibit clustering of CD36 up to around 6 h after infection, however, by 24 h (B) some PVs presented accumulation of CD36 (arrows), probably associated with the differentiation to amastigote forms. Bar: 5 μm.</p

Lentiviral expression of mCherry-<i>CD36</i> in <i>CD36</i>^-/- iMOs rescues the small PV phenotype and shows the recruitment of CD36 to PVs.

<p>(A) <i>CD36</i>-deficient iMOs exhibited smaller PVs than WT cells at 48 h of infection. The expression of mCherry-CD36 via a transducer lentiviral expression vector, restored the WT PV size to the <i>CD36</i>^-/- iMOs. A representative result of 2 independent experiments is shown, n = 200. Statistical significance was determined by Dunn's multiple comparison test. (B-D) <i>L</i>. <i>amazonensis</i> amastigotes induced clustering of CD36 at the cell membrane and in the PV. (B) Clusters of CD36 present in the cell membrane at the region of contact with an extracellular amastigote and in the PV of another intracellular parasite (arrows). (C) By 24 h after infection, the clusters of CD36 are always found in the PV membrane where the parasite is anchored by its posterior pole (arrow). (D) Formaldehyde fixed amastigotes also induce the clustering of CD36 in the PV (arrow). Bar: 5 μm. (C-D) At least 25 parasites were analyzed in 2 independent assays.</p

<i>CD36</i>^-/- BMDMs have normal PV acidification and reduced lysosomal fusion to the PVs.

<p>(A) Acidification of PV was normal in <i>CD36</i>^-/- macrophages. The pH was about 4.5 at 15 min of infection and stabilized at pH 4.0 at 1 h of infection in WT and CD36-deficient cells. The results shown are an average of 3 independent experiments. (B) Twenty four hours post-infection macrophages were loaded with a 15 min pulse of pHrodo Dextran and the accumulation of this tracer in each PV was measured by confocal microscopy after a 15 min chase. CD36-deficient PVs exhibited reduced accumulation of pHrodo coming from the endocytic pathway (t-test, n = 100). (C-E) Macrophages were infected and immunostained with anti-LAMP1/2 antibodies, followed by fluorescence microscopy imaging and quantitation. (D) C57BL/6 and <i>CD36</i>^-/- macrophages had comparable percentages of LAMP positive PVs at early time points, but LAMP proteins gradually accumulated significantly more in the PVs from WT BMDMs at 48 h and 72 h post-infection. (E) The total fluorescence of LAMP1/2 is comparable between WT and CD36-deficient macrophages. Mean data of 300 cells imaged in 2 independent experiments. (F) To assess lysosomal protease activity, Cathepsin B activity was determined by measuring the hydrolysis of the synthetic substrate Z-RR-pNA. Lysosomal protease activity was normal in CD36-deficient macrophages indicating that CD36-deficient cells are not defective in lysosome biogenesis. (G-H) Macrophages were infected for 24 h and stained with Lysotracker. (G) Uninfected and 24 hour infected macrophages showing lysotracker signal (green), the arrows indicate typical Lysotracker positive organelles scored in the assay and the arrow heads indicate the amastigotes. (H) Lysotracker positive organelles were quantified in complete Z-stacks. WT cells lose lysosomes as they fuse with PV after parasite infection, while uninfected <i>CD36</i>^-/- macrophages exhibited lower Lysotracker positive organelles which remain unaltered after infection. Results from a pool of 2 independent experiments with at least 27 cells in total, t-test.</p